Core D provides technical expertise and state of the art molecular and cellular biology methodology for[unreadable] cloning and expression of the proteins described in the program project. This centralized operation coordinates[unreadable] all molecular biology work and ensures that all proteins are cloned and expressed in a uniform[unreadable] fashion. This is particularly important since several investigators plan to analyze different aspects of some of[unreadable] the same proteins. This uniformity greatly enhances accuracy of comparison between the proteins.[unreadable] Core D designs and clones expression vectors encoding wild type or mutated proteins as requested by the[unreadable] individual investigators. All wild type genes described in this program project are identified, sequenced and[unreadable] assembled in cloning vectors. Thus, vectors expressing wild type or mutated proteins can be cloned from[unreadable] cDNAs. Vectors expressing consensus sequence peptides will be constructed from synthetic[unreadable] oligonucleotides. Depending on the requirement for the analysis of the protein (amount and/or modification of[unreadable] protein), the expression vectors are designed for expression in prokaryotic or eukaryotic cell lines. Core D[unreadable] also establishes cell lines expressing the proteins, determines conditions for optimum protein expression and[unreadable] aids in the purification of the expressed protein.[unreadable] Thus the service of Core D includes: a) advice toward an optimal expression system, b) design[unreadable] methodologies to express desired proteins, c) confirm wild type DNA sequence by restriction and/or DNA[unreadable] sequence analysis, d) design primers for DNA sequence analysis, e) design primers for PCR amplification of[unreadable] wild type or mutant constructs, f) extract DNA, g) digest DNA with restriction enzymes, h) amplify DNA by[unreadable] PCR, i) ligate DNA (fragments) in cloning and expression vectors, j) transform/transfect ligated DNA into cell[unreadable] lines to express the proteins, k) select clones, I) amplify clones, m) isolate permanently transfected cell lines,[unreadable] n) maintain transformed cell lines, o) establish optimum protein expression conditions, p) assist in protein[unreadable] purification, q) maintain cell lines carrying wild type and expression vectors.[unreadable] All personnel in Core D trains new users associated with the projects in molecular biology and cell culture[unreadable] techniques and oversees proper use of cell culture facilities. This ensures safe maintenance of cell systems[unreadable] and guarantees compliance with NIH guidelines.[unreadable] Core D designs and clones expression vectors encoding proteins requested by the principal investigators of[unreadable] the individual projects. They establish cell lines, determine optimum conditions for protein expression and aid[unreadable] in the purification of the expressed protein.[unreadable]